The present invention provides a method for determining the nucleotide sequence of DNA molecules (referred to herein as the nucleotide base sequence or simply the base sequence). Several methods are known for sequencing DNA molecules such as methods of F. Sanger, S. Nicklen, A. R. Coulson, Proc. Natl. Acad. Sci. U.S.A., 74, 5463 (1977), and A. M. Maxam and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A., 74, 560 (1977). These known methods use various means for producing labeled fragments of DNA, each of which terminates with a known base (A, G, C or T). These fragments are then separated by length, typically by an electrophoretic gel, utilizing a different gel strip for each type of terminal base. The DNA sequence is then read from the gel strips. As a variation, instead of using the same label for each fragment (such as a fluorescent dye or radioactive label) J. M. Prober, et al., Science, 238, 336-341, Oct. 1987, and C. Connell et al., BioTechniques, Vol. 5, No. 4, 342-348 (1987), use a different dye to label each of the different base termination fragments so there is a different dye associated with A, G, C and T termination. This modification allows a single gel to be used, however, it also introduces new problems due to the effect of the different dyes on fragment mobility.
A limitation of the prior methods is that they are apparently limited by the rate at which the fragments may be separated and are also limited by the number of bases that can be sequenced in a given run by the resolution obtainable on the gel. The separation rate is inherently limited, for example, by thermal distortion of the gel caused by electrical heating, and thus the identification can only be obtained as often on average as about a few bases per minute. Also the resolution on the gel is a maximum of about 1,000 bases, with improvement in this resolution not being likely because of band compression effects, and because there are interactions between the DNA strands which dominate over the length effect of very long strands, thus confusing the signal for long fragments.
The present invention provides an improvement over these prior art methods.
It is thus an object of the present invention to provide a method of DNA identification in which the rate limiting step is essentially the rate of a polymerase reaction, which is usually on the order of at least 60 bases per second, or limited by the rate in which the reagents can be delivered to the reaction site, whichever is slower.
It is another object of the present invention to provide a method of DNA sequencing in which the accuracy does not depend upon the length of the DNA molecule to be sequenced but, rather on the signal-to-noise ratio of the detection means, which is very low using optical detection methods. Such high sensitivity detection means provide the advantage that only very small quantities of DNA are necessary, typically, less than a million molecules.
It is yet another object of the present invention to provide a DNA sequencing which is unambiguous even in short sequences of identical bases, which are difficult to distinguish by prior art methods.
Another object of the present invention is to provide a novel method for DNA sequencing in which the reagents for detection comprises a single mixture of bases, and does not require four separate preparations (one for each base) as required by methods of the prior art.
These and other objects of the present invention will be apparent from the following description, the appended claims and from practice of the invention.